Cell culture and biochemical procedures were used to identify and study the possible mechanisms regulating the secretion of GnRH-like immunoreactivity (GnRH-LI) from human placenta. Monolayer primary cultures of trophoblasts were established after mechanical and enzymatic dispersion of normal human term placenta. The cultured cells stained immunocytochemically positive with anti-GnRH serum, and GnRH-LI extracted from the cells eluted from high performance liquid chromatography with the same retention time as authentic GnRH. One week after plating, exposure to high concentrations of K+ or to various doses of veratridine, a Na+ ionophore, increased GnRH-LI release into the culture medium. This effect was reversed by Ca2+ antagonists (cobalt, EGTA, and verapamil). Dibutyrylcyclic AMP, forskolin, theophylline, and theobromine also increased GnRH-LI concentrations in the medium of cultured placental cells in a dose-related manner, as did prostaglandins E2 and F2 alpha and epinephrine. The effect of epinephrine on GnRH-LI concentrations was mimicked by isoproterenol and reversed by propranolol, suggesting an effect mediated by beta-adrenergic receptors. These results indicate that GnRH-LI release from cultured human placental cells is stimulated by the opening of ionic channels and activation of the adenylate cyclase/cAMP system, and that prostaglandins and epinephrine may be involved in the regulation of GnRH-LI release from human placenta.